Hi,
I'm going to be building a gene from scratch come this fall, using
oligos, and am hoping to collect thoughts, examples and advice.
My gene is 350 codons, and comes from a ciliated protozoa, so uses a
non-standard genetic code (use of TAA TAG "stops" for Gln). I'll rebuild
the gene to replace some 15 stops, as well as improve the codon usage for
expression in E. coli, and perhaps insect cells (though it's not clear to
me at the moment how one goes about optimizing codon usage for two
species). And are there programs for introducing silent restriction
sites?
I have this vision of having ~20 oligos made, mixing them all, annealing
slowly, adding ligase and cloning to an expression vector. A day's work!
But seriously, how does one put them together, does it have to be in
steps, how much effort should be put into confirming correct
intermediates, as opposed to just screening the final product?
How many issues have I not even considered?
Thanks so much,
Tom Doak
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Department of Oncological Sciences
Division Molecular Biology and Genetics
U of Utah, School of Medicine, Rm. 5C334
SaltLakeCity, UT 84132
801-581-3747
tdoak at genetics.utah.edu